Non-healing wounds are a major health care problem, and at any given time more that 3 million Americans have chronic wounds. The overall objective of this research plan is to gain a better understanding of the molecular and cellular effect of growth factors on would repair. This research proposal is based on the hypothesis that wound repair can be accelerated if one can: 1) specifically deliver certain growth factor genes into the wound microenvironment; 2) optimize their level of expression; and 3) optimize the timing of expression. Wounds in aged pigs are studied because pig skin is similar to human skin and delayed or absent healing is more common in the elderly. This research plan contains a combination of three new concepts for promoting wound healing. First is the use of a sealed chamber to cover the wound, creating an incubator environment. With use of this model, the wound micro-environment can readily be modified in a specific fashion like a tissue culture in vivo. Secondly, the wound healing process is modulated by targeting delivery of growth factor genes into the wound microenvironment using in vivo microseeding gene transfer. This method yields much higher levels of gene expression than gene transfer mediated by particle-bombardment or single injection. Because gene transfer offers targeted local and persistent delivery of therapeutic polypeptides, a single genetic intervention should be sufficient to adequately promote wound repair. Thirdly, as roles of different growth factors may differ at various stages of the healing process and uncontrolled expression of growth factors may delay healing, the production of exogenous growth factors in the wound will be turned on and off with a tetracycline-regulated genetic switch. With this switch, the timing and therapeutic levels of growth-promoting gene expression can be precisely controlled, maximizing the biological effect of the growth factors delivered. Therefore, the expression of exogenous growth factors can be easily regulated to complement the endogenous profile. Moreover, with a switch that allows precise timing of expression, the role of specific growth factors at each individual stage of healing can be elucidated. In Specific Aim One, we will attempt to increase the rate of healing by up- regulating growth factors in the wound environment by microseeding gene transfer. The Second and Third Specific Aims will combine in vivo and ex vivo gene transfer with the tetracycline-reversible genetic switch developed in our laboratory. When proven effective in pigs, these methodologies could be used in patients with non-healing wounds. To our knowledge this is also the safest system available for in vivo and ex vivo gene delivery to skin, offering therapeutic potential for other skin diseases.